Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models
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PROVIDER: PRJNA742072 | ENA |
REPOSITORIES: ENA
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Project description:Type 2 diabetes mellitus is one of the most prevalent metabolic diseases affecting multiple organs, including reproductive disorders in male diabetic patients. However, the molecular mechanisms that contribute to spermatogenesis dysfunction in diabetic patients have not yet been fully elucidated. Here, we performed Smart-seq2 to examine the transcriptome of diabetic patients' testis cells at single cell resolution including all major cell types of the testis. Intriguingly, whereas spermatogenesis appears largely preserved, the gene expression profiles of Sertoli cells and the blood-testis barrier (BTB) structure were dramatically impaired. Among the deregulated pathways, the Apelin (APLN) peptide /Apelin-receptor (APJ) axis was hyper-activated in diabetic patients. Mechanistically, APLN is produced locally by Sertoli cells upon high glucose treatment, which subsequently suppressed the production of carnitine and repressed the expression of cell adhesion genes in Sertoli cells. Together, these effects culminated in BTB structural dysfunction. Finally, using the small molecule APLN receptor antagonist, ML221, we show that blocking APLN/APJ significantly ameliorated the BTB damage and, importantly, improved functional spermatogenesis in diabetic db/db mice. We also translated and validated these findings in cultured human testis. Our findings identify the APLN/APJ axis as a promising therapeutic target to improve reproduction capacity in male diabetic patients.
2022-10-27 | GSE179080 | GEO
Project description:Type 2 diabetes mellitus is one of the most prevalent metabolic diseases presenting with systemic pathologies, including reproductive disorders in male diabetic patients. However, the molecular mechanisms that contributing to spermatogenesis dysfunction in diabetic patients have not yet been fully elucidated. Here, we perform STRT-seq to examine the transcriptome of diabetic patients' testes at single-cell resolution including all major cell types of the testis. Intriguingly, whereas spermatogenesis appears largely preserved, the gene expression profiles of Sertoli cells and the blood-testis barrier (BTB) structure are dramatically impaired. Among these deregulate pathways, the Apelin (APLN) peptide/Apelin-receptor (APJ) axis is hyper-activated in diabetic patients' testes. Mechanistically, APLN is produced locally by Sertoli cells upon high glucose treatment, which subsequently suppress the production of carnitine and repress the expression of cell adhesion genes in Sertoli cells. Together, these effects culminate in BTB structural dysfunction. Finally, using the small molecule APLN receptor antagonist, ML221, we show that blocking APLN/APJ significantly ameliorate the BTB damage and, importantly, improve functional spermatogenesis in diabetic db/db mice. We also translate and validate these findings in cultured human testes. Our findings identify the APLN/APJ axis as a promising therapeutic target to improve reproduction capacity in male diabetic patients.
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Project description:Dysregulated APLN have been widely reported in tumorigenesis, yet the role of the proteolytic cleavage of APLN precursor proAPLN in oncogenesis is not known. Here, we identify Furin the unique proprotein convertase able to cleave proAPLN in cancer cells. We generated for preclinical development APLN-DM peptide following alteration of proAPLN cleavage sites. The proteomic analysis was designed to identify dysregulated proteins in APLN and APLN-DM-expressing cells.
2025-01-15 | PXD045256 | Pride
Project description:We used bulk RNAseq to analyze crypt intestinal epithelial gene expression changes between wild-type and Apln-/- adult mice
2022-03-29 | GSE178110 | GEO
Project description:In epithelia, a primary damage of tight junctions (TJ) always leads to a secondary disruption of adherens junction (AJ), and vice versa. This response, if occurring in the testis, would disrupt spermatogenesis because the blood-testis barrier (BTB) must remain intact during the transit of spermatids in the seminiferous epithelium, which is associated with extensive apical ectoplasmic specialization (apical ES, a testis-specific AJ type) restructuring. As such, apical ES restructuring accompanied with the transit of developing spermatids during spermiogenesis must be segregated from the BTB to avoid an immunological barrier breakdown in all stages of the seminiferous epithelial cycle, except at stage VIII when spermiation and BTB restructuring take place concurrently. We report herein a mechanism involving restricted spatial and temporal expression of Arp2/3 complex and N-WASP, whose actin branching activity associated with apical ES and BTB restructuring in the seminiferous epithelium. High expression of Arp3 at the apical ES was shown to correlate with spermatid movement and proper spermatid orientation. Likewise, high Arp3 level at the BTB associated with its restructuring to accommodate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. These findings were validated by in vitro and in vivo studies using wiskostatin, an inhibitor that blocks N-WASP from activating Arp2/3 complex to elicit actin branching. Inhibition of actin branching caused a failure of spermatid transit plus a loss of proper orientation in the epithelium, and a "tightened" Sertoli cell TJ permeability barrier, supporting the role of Arp2/3 complex in segregating the events of AJ and BTB restructuring.
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Project description:The mechanism(s) that regulate and coordinate the events of spermiation and blood-testis barrier (BTB) restructuring in the seminiferous epithelium that occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis are unknown. In this report, fragments derived from the laminin complex composed of laminin alpha3, beta3, and gamma3 chains (laminin-333) at the apical ectoplasmic specialization (apical ES) were shown to modulate BTB dynamics directly and/or indirectly via hemidesmosome. Experiments were performed using cultured Sertoli cells with functional tight junction (TJ) barrier and the ultrastructural features of the BTB but not apical ES. Recombinant protein fragments of laminin beta3 and gamma3 chains were shown to reduce the protein levels of occludin and beta1-integrin dose dependently at the Sertoli-Sertoli and Sertoli-basement membrane interface, respectively, thereby destabilizing the BTB permeability function. These results were corroborated by transient overexpression of laminin fragments in Sertoli cells. To further assess the role of beta1-integrin in hemidesmosome, knockdown of beta1-integrin in Sertoli cells by RNAi was found to associate with occludin redistribution at the Sertoli-Sertoli cell interface, wherein occludin moved away from the cell surface and became associated with endosomes, thereby destabilizing the BTB. In short, an apical ES-BTB-hemidesmosome autocrine regulatory axis was identified in testes, coordinating the events of spermiation and BTB restructuring that occur at the opposite ends of the seminiferous epithelium during spermatogenesis.
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Project description:Deoxynivalenol (DON) is among the most prevalent contaminants in cereal crops and has been demonstrated to impair male spermatogenesis and induce oxidative stress, testicular apoptosis, and disruption of the blood-testis barrier (BTB). Lactoferrin (LF) is an iron-binding glycoprotein with multifunctions including anti-inflammation and antioxidation. Thus, this study aimed to investigate the effects of LF on the spermatogenesis and integrity of the BTB in DON-exposed mice. Thirty-two male mice were allotted to four groups for a 35-day feeding period: vehicle (basal diet), DON (12 mg/kg), LF (10 mg/d, p.o.), and DON + LF. The results showed that DON induced vacuolization of the spermatogenic epithelium, broke the adhesion junction between Sertoli cells and spermatids established by N-cadherin and induced testicular oxidative stress. LF administration restored sperm production, attenuated the DON-induced oxidative stress and reduced the breakages in adhesion junction. DON exposure enhanced the protein expression of occludin. Transcriptional profiling of the testis observed a disturbance in the expression profiles of cell adhesion and inflammatory response genes, and LF administration reversed these gene expressions. Furthermore, down-regulated signaling pathways, including the apical junction, TNFα signaling via NF-κB, and TGF-β in the DON group were observed. These were restored by LF. Enrichment analysis between DON + LF group and vehicle also confirmed the absence of these pathways. These findings indicated that LF eliminated the DON-induced detriment to spermatogenesis and cell connections between Sertoli cells and spermatids via improving antioxidant capacity and modifying the inflammatory response and cell adhesion genes.
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Project description:About 10% of couples of reproductive age worldwide suffer from infertility, with male factors accounting for about 50%, and testosterone is essential for male fertility, but the mechanisms behind the poor sperm quality and abnormally low blood testosterone levels in diabetic patients are unclear. In this study, we found that the peptide Apelin (APLN) significantly inhibited testosterone secretion from Leydig cells (LCs), which in turn led to decreased sperm quality. APLN was found to be localized to leydig cells in the testes of several diabetic mice, and its expression was significantly increased. The addition of APLN to mouse leydig cells decreased testosterone content and decreased the expression of genes related to testosterone synthesis, but the addition of ML221, an APLN receptor inhibitor, significantly increased the expression of testosterone synthesis genes. Injecting ML221 into diabetic mice significantly increased testosterone levels in the blood, increased sperm count as well as enhanced sperm motility and ultimately increased live birth rate of mice after in vitro fertilization. Proteomics revealed that ML221 significantly increased the expression of NR2F2 and further activated the transcription of testosterone synthesis genes. This study will further use mouse and cellular models to completely unravel the specific mechanisms by which APLN inhibits testosterone secretion and elucidate the causes of decreased sperm quality in diabetic patients. This study provides a new clinical target for the development of drugs to improve fertility in diabetic patients.
2025-02-24 | GSE289564 | GEO
Project description:About 10% of couples of reproductive age worldwide suffer from infertility, with male factors accounting for about 50%, and testosterone is essential for male fertility, but the mechanisms behind the poor sperm quality and abnormally low blood testosterone levels in diabetic patients are unclear. In this study, we found that the peptide Apelin (APLN) significantly inhibited testosterone secretion from Leydig cells (LCs), which in turn led to decreased sperm quality. APLN was found to be localized to leydig cells in the testes of several diabetic mice, and its expression was significantly increased. The addition of APLN to mouse leydig cells decreased testosterone content and decreased the expression of genes related to testosterone synthesis, but the addition of ML221, an APLN receptor inhibitor, significantly increased the expression of testosterone synthesis genes. Injecting ML221 into diabetic mice significantly increased testosterone levels in the blood, increased sperm count as well as enhanced sperm motility and ultimately increased live birth rate of mice after in vitro fertilization. Proteomics revealed that ML221 significantly increased the expression of NR2F2 and further activated the transcription of testosterone synthesis genes. This study will further use mouse and cellular models to completely unravel the specific mechanisms by which APLN inhibits testosterone secretion and elucidate the causes of decreased sperm quality in diabetic patients. This study provides a new clinical target for the development of drugs to improve fertility in diabetic patients.
2025-02-24 | GSE289563 | GEO
Project description:Cultivation of neonatal mouse testis tissue can induce spermatogenesis and produce fertile sperms. However, this in vitro spermatogenesis by the current organ culture method comes short in fully mimicking the in vivo counterpart, partly due to a lack of knowledge about the underlying molecular phenotypes. In this study, we investigated transcriptome of cultured testis tissues using microarray method. Principle component analysis of the transcriptome data revealed delay and/or arrest of spermatogenesis and immediate radical immune reactions in the cultured testis tissues. The delay/arrest of spermatogenesis occurred before and during early meiotic phase, resulting in inefficient progression of meiosis. The immune reaction, on the other hand, was drastic and overwhelming, in which TLR4-NF-kB signaling was speculated to be involved. Notably, treatment with TAK242, an inhibitor of TLR4-NF-kB signaling pathway, ameliorated the macrophage activation which otherwise would exacerbate the inflammation. The present study revealed for the first time the immense immune reaction in the cultured tissue by transcriptomic analysis, which could be a cause of the inefficiency of in vitro spermatogenesis.
2020-06-30 | GSE147982 | GEO